P1403: ENDOMUCIN IS A MARKER OF QUIESCENT HAEMATOPOIETIC STEM CELLS WITH HIGH LONG-TERM ENGRAFTMENT CAPACITY
Authors: S. Engelhard, M. Drilsvik, F. G. Rodriguez Gonzalez, C. Nerlov, J. Weischenfeldt, K. Reckzeh, K. Theilgaard-Mönch
Journal: HemaSphere (2022)

Background: The purification of haematopoietic stem cells (HSCs) is crucial for studies on normal and leukaemic stem cell biology. We recently identified Endomucin (EMCN) as a novel HSC marker, a transmembrane protein conserved in mouse and human throughout development, but not in lineage-committed haematopoietic progenitor cells. Therefore, EMCN can be potentially used in combination with “classic” HSC markers to refine the immuno-phenotype, thus improving HSC purification for studies in HSC biology. Aims: Our aim was to characterise EMCN as a novel complementary marker for purification of HSCs and to investigate its biological function in an EMCN knock-out (KO) mouse model. Methods: Using flow cytometry, we sub-classified HSCs by combining “classic” HSC markers (CD150+CD48-LSK) with EMCN into EMCN+ HSCs and EMCN- HSCs. We characterised these populations (i) functionally by transplantation and in vitro assays, molecularly by (ii) RNA-sequencing and by (iii) flow cytometry-based cell cycle analysis. We further analysed “classic” HSCs molecularly and functionally in EMCN KO mice. Results: Gene set enrichment analysis (GSEA) indicated that EMCN+ HSCs are more quiescent than EMCN- HSCs, since gene signatures associated with DNA replication were upregulated in the EMCN- HSCs vs EMCN+ HSCs. Consistently,EMCN+ HSCs demonstrated upregulation of RhoGTPase signatures associated with low migration activity. Accordingly, genes associated with mitochondrial biogenesis were upregulated in EMCN- HSCs as compared to EMCN+ HSCs, indicating that these cells are metabolically more active. In addition, EMCN- HSCs demonstrated upregulation of genes regulated by Runx1, a key transcription factor involed in haematopoietic differentiation, highlighting that EMCN- HSCs are primed for proliferation and differentiation. Thus, our data suggest that EMCN expression on HSCs is associated with quiescence and reduced migration potential, which are characteristics of HSCs residing in the stem cell niche. Flow cytometry analysis showed that EMCN+ HSCs were more quiescent as compared to EMCN- HSCs (G0: p = 0.001; G1: p < 0.0001; S-G2M: p = 0.014; n = 10), and single cell cultures demonstrated that EMCN+ HSCs initiate their first division in vitro later than EMCN- HSCs (undivided cells after 2 days- EMCN+ HSCs: 18.6% vs EMCN- HSCs: 2.6%; p = 0.04, n= 3). Consistent with the more quiescent phenotype of EMCN+ HSCs, limiting dilution transplantation experiments showed that EMCN+ HSCs contained a higher frequency of long-term repopulating HSCs as compared to EMCN- HSCs (estimated HSC frequency according to limiting dilution analysis: EMCN+ 1 in 7, EMCN- 1 in 42; p < 0.001). Functional and molecular characterisation of HSCs in EMCN KO mice revealed no effect of the loss of EMCN on HSC function during steady-state haematopoiesis or multi-lineage bone marrow reconstitution after two rounds of serial transplantation. Summary/Conclusion: In conclusion, EMCN can be used in combination with “classic” HSC markers (CD150+CD48-LSK) to enrich for quiescent HSCs with high long-term engraftment capacity. However, functionally, EMCN seems to be dispensable for steady-state haematopoiesis in adult mice, and its loss does not affect the long-term engraftment capacity of HSCs in mice.